microRNAs (miRNAs) are an abundant class of small RNAs that derive from distinctive hairpin precursor transcripts. Their biogenesis typically involves cleavage by the Drosha and Dicer RNase III enzymes, and the resultant mature miRNAs mediate extensive networks of post-transcriptional regulation. In the course of analyzing the miRNA pathway in Drosophila, we uncovered atypical miRNAs derived from short hairpin introns that we term mirtrons. Their biogenesis substitutes a seemingly obligate processing event by the Drosha RNase III enzyme with the splicing machinery. We characterized the biogenesis and evolutionary range of mirtrons across four animal species, using biochemical, molecular, genetic, and computational methods. This work led to a series of unexpected avenues that further expanded the diversity of non- canonical miRNAs, including a hybrid pathway that marries splicing with the RNA exosome to generate hairpins that serve as Dicer substrates, as well as short hairpins that generate miRNAs in a Dicer-independent mechanism in vertebrates. In addition, this work served as a foundation of our biological studies of miRNA functions. For our renewal application, we selected several directions on understanding enzymes and mechanisms that mediate the biogenesis of mirtrons and Dicer-independent miRNAs. We present extensive unpublished data for our proposed Aims, including the discovery of hundreds of mammalian mirtrons, including a novel 3' tailed subtype in mammals, the first demonstration of Dicer-independent miRNA function in Drosophila, and establishment of in vitro assays for Dicer-independent miRNA production. These preliminary data are the basis of individual gene studies to understand detailed mechanisms, which we will extend with genomewide perspectives. In addition, we wish to relate these findings to their broader implications with respect to other small RNAs (i.e. canonical miRNAs) and discovery of novel substrates. Finally, we are concerned with relating our research to larger evolutionary questions, including the evolutionary emergence of miRNAs and of RNAi itself, and with exploiting our mechanistic knowledge to advance RNA silencing as a technological tool.